Cyclic di-AMP-mediated interaction between Mycobacterium tuberculosis ΔcnpB and macrophages implicates a novel strategy for improving BCG vaccination

Cyclic di-AMP (c-di-AMP) has been shown to play an important role in bacterial physiology and pathogen-host interactions. We previously reported that deletion of the sole c-di-AMP phosphodiesterase-encoding gene (cnpB) in Mycobacterium tuberculosis (Mtb) led to significant virulence attenuation. In this study, we found that Delta cnpB of M. bovis bacillus Calmette-Guerin (BCG) was unable to secrete c-di-AMP, which differs from Mtb Delta cnpB. We infected bone marrow-derived macrophages (BMDMs) with c-di-AMP-associated mutants generated from both Mtb and BCG. Our results showed that upon infection with Mtb Delta cnpB, BMDMs of wildtype mice secreted a large amount of interferon-beta (IFN-beta) post-infection similarly as we reported previously. In contrast, the response was less pronounced with BMDMs isolated from cGAS(-/-) mice and was nearly abolished with BMDMs prepared from STING(-/-) mice. Deletion of the region of difference 1 (RD1) locus in Mtb Delta cnpB did not alter the c-di-AMP secretion of Delta cnpB but eliminated the IFN-beta production in the infected cells. In contrast, neither BCG Delta cnpB nor a recombinant BCG Delta cnpB with a pRD1 cosmid induced a type I interferon response. Interestingly, multiple studies have demonstrated that type I IFN enhances BCG's immunity. Thus, amending BCG based on our findings might improve BCG vaccination.